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Title: | C型肝炎病毒非結構蛋白質5A高度磷酸化需要非結構蛋白質3的自我切割以及與三磷酸腺苷結合之能力 Both Auto-Proteolytic and ATP-Binding Activities of Hepatitis C Virus Non-Structural Protein 3 are Required for Non-Structural Protein 5A Hyper-Phosphorylation |
Authors: | Yen-Ling Lai 賴彥伶 |
Advisor: | 余明俊(Ming-Jiun Yu) |
Keyword: | C型肝炎,非結構蛋白質3,非結構蛋白質5A,磷酸化,自我切割,與三磷酸腺?結合之能力, HCV,Non-structural protein 3,Non-structural 5A,phosphorylation,auto-proteolytic,ATP-binding activitiy, |
Publication Year : | 2016 |
Degree: | 碩士 |
Abstract: | C型肝炎病毒(Hepatitis C virus)的非結構蛋白質5A( non-structural protein 5A, NS5A)是一個磷酸化蛋白質並且有著兩種磷酸化態,包含:低度(Hypo)以及高度(Hyper)磷酸化態,其中高度磷酸化的NS5A在病毒的生活史中扮演重要的角色。先前研究已指出,NS5A的高度磷酸化會受到非結構蛋白質3( non-structural protein 3, NS3)的影響,然而NS5A上哪些位點會受到NS3的影響至今未知。我的研究目標是利用我們實驗室所擁有的專一性抗體針對S2198、S2208、S2211和S2214的磷酸化,來探討NS5A上哪些磷酸化位點會受到NS3的影響。我們利用突變的方式扼殺了NS3蛋白酶(protease)的活性,結果顯示多蛋白(polyprotein)無法被切割,而且NS5A上S2208、S2209和S2211這三個位點皆無法被磷酸化。另外,這三個位點的磷酸化以及NS3和4A間的切割無法用蛋白酶功能正常的NS3來補救,顯示NS3和4A之間的切割對於NS5A的高度磷酸化是必要的。接著,我們進一步利用點突變的方法阻擋NS3、4A、4B和5A之間的切割,發現只要NS3和4A之間無法進行正常的切割,NS5A就無法有高度磷酸化。因此我們可以推論NS5A高度磷酸化依賴同分子上NS3和4A之間的自行切割。這個結果也驅使我們研究NS3與三磷酸腺苷(ATP)的結合活性是否會影響NS5A的高度磷酸化。結果顯示,當我們利用突變去降低NS3與ATP的結合能力時,NS5A上的S2208、 S2211和S2214的磷酸化程度大幅降低,由此可以推論出NS5A高度磷酸化需要NS3的自行切割能力以及與ATP的結合活性。 The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein required for the viral life cycle. It has hypo- and hyper-phosphorylation states; the latter has been implicated in viral replication and assembly. NS5A hyper-phosphorylation is influenced by the non-structural protein 3 (NS3); however, the exact phosphorylation sites affected by NS3 are unknown. In this thesis, I used four phospho-specific antibodies to NS5A S2198, S2208, S2211 and S2214 to study how NS3 participates in NS5A hyper-phosphorylation. Mutation at the NS3 catalytic serine residue resulted in an un-cleaved NS3-5A polyprotein that was not phosphorylated at S2208, S2211 or S2214. This defect in phosphorylation could not be rescued by providing an active NS3 in trans, suggesting that NS5A phosphorylation at these sites requires cleavage of the polyprotein by NS3 in cis. Mutations that block cleavage at the NS3-4A junction, not the NS4A-4B or NS4B-5A junction, abolished S2208, S2211 and S2214 phosphorylation. These results suggest that NS5A hyper-phosphorylation depends on successful intramolecular auto-cleavage at the NS3-4A junction and prompted us to examine whether ATP-binding ability of NS3 affects NS5A hyper-phosphorylation. Indeed, mutations that reduce NS3 ATP-binding ability reduced NS5A hyper-phosphorylation at S2208, S2211 and S2214. We conclude that auto-proteolytic and ATP-binding activities of NS3 are required for NS5A hyper-phosphorylation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19304 |
DOI: | 10.6342/NTU201601076 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 生物化學暨分子生物學科研究所 |
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ntu-105-1.pdf Restricted Access | 2.93 MB | Adobe PDF |
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